DE SCRIPTION (provided by applicant): The major cause of disease in cystic fibrosis (CF) is the inability of the protein encoded by the CF gene, CFTR, to achieve the native, folded state that is permitted to traffic to the apical membranes of epithelial cells. The absence of CFTR at this site results in insufficient airway surface liquid, impairing the clearance of mucins and bacteria, and leading to recurrent infection, inflammation and bronciectasis. CF provided an early model for the growing list of 'conformational diseases' in which defects in protein folding, trafficking and aggregation contribute to a diverse set of pathologies. Several components of the machinery controlling CFTR biogenesis have been identified, including chaperones and co-chaperones that facilitate CFTR biosynthesis, and proteins that target unfolded CFTR intermediates for polyubiquitination and proteasomal degradation. We recently identified a new CFTR co-chaperone with two functions in CFTR biogenesis: the cysteine string protein (Csp) acts as a co-chaperone to enhance CFTR biosynthesis, and it also modulates CFTR exit from the endoplasmic reticulum (ER). Csp is a J-domain protein that binds and activates Hsc70, a major CFTR chaperone. Csp is expressed in the ER of epithelial cells, where it interacts physically with immature CFTR by binding to the N-terminus and R domain with an affinity much higher than other chaperones. These findings provide the basis for our hypothesis, that Csp is an integral component of the machinery that controls CFTR biosynthesis and ER export. This proposal is designed to determine how the domain-specific, high-affinity interactions of CFTR with Csp contribute to CFTR biogenesis, and where these interactions fail in the processing of the common CFTR folding mutant, deltaF508. To achieve these goals, we will evaluate the interactions of two co-chaperones, Csp and Hdj-2, with CFTR during its biosynthesis. Structure-function studies will identify the sites of high-affinity interactions between Csp and CFTR (relative to Hdj-2 binding), and the significance of these interactions will be evaluated using CFTR folding assays. The presence of Csp in pre-budding complexes and its control of CFTR budding from the ER will indicate how Csp interactions with CFTR and other proteins influence ER export. Finally, we will determine the role of CFTR and Csp phosphorylation in CFTR biogenesis and ER exit, specifically, the concept that their phosphorylation alters protein-protein interactions to regulate these processes. The co-chaperone and chaperone interactions that govern the CFTR protein processing are not resolved. More than 90% of CF patients have the common deltaF508 folding mutant on at least one allele, so that amelioration of this mutant's processing defect represents a reasonable therapeutic target. Understanding the role of direct, high affinity interactions with Csp may identify targets for favorable and selective modulation of CFTR biogenesis.